The overall objectives of this project are to gain insight into the signal transduction mechanisms of the growth factor thrombopoietin (TPO) and its receptor, Mpl, and to provide in-depth training in molecular biology for Dr. Debra Dahlen. This career development award will enable Dr. Dahlen to achieve her immediate goal of exploring the role of Mpl in stem cell proliferation and differentiation and her long-term career goal of becoming an independent clinician-scientist involved in laboratory- based research affiliated with a major academic medical center. To achieve these goals, Dr. Dahlen will follow a research career development plan consisting of a program of didactic sessions and a laboratory- based research project under the sponsorship of Dr. Jonathan G. Drachman and under the consulting guidance of Drs. Jan Abkowitz, Tony Blau, Ken Kaushansky, Thalia Papayannopoulou, and Dennis Willerford. The specific aims of this research project are to: 1) determine tile receptor motifs that facilitate internalization of Mpl, and to identify the mechanism by which this occurs". 2) identify the signaling sequences necessary for normal intracellular trafficking and determine the intracellular fate of Mpl and 3) investigate the function of TPO/Mpl internalization and intracellular trafficking in cellular proliferation and megakaryocyte development/differentiation. The research plan includes site-directed mutagenesis, FACS analysis, Western blotting, subcellular fractionation, confocal microscopy, proliferation assays, signal transduction studies in cell lines and primary cells, and development of novel chimeric proteins. Although an ambitious set of experiments, the wide variety of techniques and methodology will help develop the breadth of Dr. Dahlen's scientific experience. The clinical significance of this project is threefold: 1) TP0/MpI signaling is non-redundant in normal stem cell and megakaryocyte biology; 2) Mpl expression is associated with a poor prognosis in hematologic malignancies; and 3) abnormalities in MpI signaling have been associated with several rare forms of inherited thrombocytopenia. Understanding the mechanisms of intracellular trafficking of MpI and its interaction with other signaling molecules may lead to novel approaches in the treatment of a number of hematologic and other diseases.